EP0663013B1 - Method for delivering nucleic acids into cells - Google Patents
Method for delivering nucleic acids into cells Download PDFInfo
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- EP0663013B1 EP0663013B1 EP92920321A EP92920321A EP0663013B1 EP 0663013 B1 EP0663013 B1 EP 0663013B1 EP 92920321 A EP92920321 A EP 92920321A EP 92920321 A EP92920321 A EP 92920321A EP 0663013 B1 EP0663013 B1 EP 0663013B1
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- cholesterol
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- amine derivative
- dispersion
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/64—General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
- A61K47/554—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being a steroid plant sterol, glycyrrhetic acid, enoxolone or bile acid
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/127—Liposomes
- A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
- A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers with substantial amounts of non-phosphatidyl, i.e. non-acylglycerophosphate, surfactants as bilayer-forming substances, e.g. cationic lipids
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J41/00—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring
- C07J41/0033—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring not covered by C07J41/0005
- C07J41/0055—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring not covered by C07J41/0005 the 17-beta position being substituted by an uninterrupted chain of at least three carbon atoms which may or may not be branched, e.g. cholane or cholestane derivatives, optionally cyclised, e.g. 17-beta-phenyl or 17-beta-furyl derivatives
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J9/00—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of more than two carbon atoms, e.g. cholane, cholestane, coprostane
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
Definitions
- the present invention relates to methods for facilitating the transfer of nucleic acids into cells and to a novel cationic amphiphile useful for this purpose.
- Some but not all cationic amphiphiles are known to facilitate the transfer of DNA into cells, i.e., transfection. Although the mechanism of this activity is not yet clear, it probably involves the binding of the DNA/lipid complex with the cell surface via the excess positive charges on the complex. Cell surface bound complex is probably internalized and the DNA is released into the cytoplasm of the cell from an endocytic compartment. How the released DNA moves into the nucleus is not known.
- a cationic amphiphile contains the following four important structural elements: lipophilic group - Linker bond - Spacer arm - Amino group
- the amino group is positively charged at neutral pH. It may be a primary, secondary, tertiary or quaternary ammonium group.
- the spacer arm is usually a hydrophilic, 2 to 15-atom moiety which connects the amino group to the lipophilic group via the linker bond.
- the linker bond is either an ether, ester, amide or other hydrolyzable bond.
- the lipophilic group is a hydrophobic moiety which allows the insertion of the cationic amphiphile into the membranes of the cell or liposome. It serves as an anchor for the cationic ammonium group to attach to the surface of a cell or liposome.
- US-A-4,544,545 in col.3 lines 47-51 mentions the incorporation of genetic material into liposomes, it is not obviously leading the skilled person to the use of said liposomes in a method for facilitating transfer of nucleic acid into mammalian cells because US-A-4,544,545 relates to liposomes adapted for specific organ targeting. Transfection is not disclosed in US-A-4,544,545.
- WO-A-8800824 relates to liposomes which contain positively charged lipid components adapted for ophtalmic uses in which the liposomes are supplied as droplets to the eye of the patient. The lipid derivatives are linked to the cholesterol through an oxygen or nitrogen atom.
- J. Biol. Chem., Vol. 265, N°21, 1990, pp.12404-12409 investigates the effect of cholesterol sulfate and related compounds on the fusion of Sendai virus to biological and model neutrons.
- US patent 4,958,013 shows various formulas of cholesterol modified oligonucleotides which are chemical intermediates for modified oligonucleotides that will anchor to membranes.
- N-[1-(2,3-dioleoxyloxy) propyl]-N,N,N-trimethyl ammonium chloride is the first cationic amphiphile exhibiting the activity of transfection. Its lipophilic group is a double-chain, C18:1 aliphatic group. It contains a quaternary ammonium group connected to the lipophilic group via a 3-carbon spacer arm with two ether linker bonds. Although the molecule is effective in transfection, it is not biodegradable and is rather toxic to cells.
- Another series of cationic amphiphiles used in transfection is the quaternary ammonium detergents.
- Either single chain (such as cetyltrimethylammonium bromide) or double chain (such as dimethyldioctadecylammonium bromide) detergents exhibit activity to transfect animal cells.
- the amino group in these amphiphiles is quaternary and is connected to the lipophilic group without the spacer arm or linker bonds.
- Another single-chain detergent, stearylamine contains a primary amino group connected to a single C18:0 chain without a spacer arm or linker bond. This group of amphiphiles is also toxic to the cells.
- the first group contains two C18:1 chains as the lipophilic group. Both groups contain a quaternary ammonium group, but the spacer arm structure varies. In one case, the trimethylammohium group is directly connected to the two C18:1 chains via a 3-carbon spacer arm and ester bond.
- the amphiphile, 1,2-dioleoxy-3-(trimethylammonio)propane, (DOTAP) is a close analog of DOTMA.
- the trimethylammonium group is connected via a butanoyl spacer arm to either the double-chain (for DOTB) or cholesteryl (for ChOTB) group.
- Other amphiphiles i.e.
- 1,2-dioleoyl-3-succinyl-sn-glycerol choline ester (DOSC) and cholesteryl hemisuccinate choline ester, ChOSC contain a choline moiety as the quaternary ammonium group which is connected to the double-chain (for DOSC) or cholesteryl (for ChOSC) group via a succinyl spacer arm.
- DOSC 1,2-dioleoyl-3-succinyl-sn-glycerol choline ester
- ChOSC cholesteryl hemisuccinate choline ester
- lipopolyamine is L-5-carboxyspermine which contains 2 primary and 2 secondary ammonium groups.
- the cationic group is connected to two different double-chain, C16:0 lipophilic group via an amidoglycyl (for DOGS) or phosphorylethanolamine (for DPPES) spacer arm. These compounds are especially efficient in transfecting the primary endocrine cells without cellular toxicity.
- a lipopolylysine reagent for transfection has also been reported.
- the reagent contains a polylysine moiety as the ammonium group which is connected to a phospholipid (N-glutarylphosphatidylethanolamine). Therefore, the spacer arm is the side chain of lysine and the head group of the phospholipid.
- the lipophilic group is a double-chain, C18:1 group connecting to the spacer arm via two ester bonds.
- An ideal transfection reagent should exhibit a high level of transfection activity without scraping or any other mechanical or physical manipulations of the cells or tissues.
- the reagent should be non-toxic or minimally toxic at the effective doses. It should also be biodegradable to avoid any long-term adverse side-effects on the treated cells.
- reagents which fulfill these criteria contain a linker bond that is hydrolyzable in the cell.
- DOBT and DOSC both contain ester linker bonds
- cationic amphiphiles containing ester linker bonds are not stable when stored in an aqueous solution. This is probably due to a base-catalyzed hydrolysis reaction mediated by the amino group of the amphiphile.
- PKC protein kinase C
- the present inventors have therefore synthesized a series of novel cationic amphiphiles and screened their activities to inhibit PKC.
- Several amphiphiles which exhibit weak inhibitory activities towards PKC are particularly suitable for transfections.
- the stability of the bond in aqueous solution is much greater than that of the ester bond, yet it is hydrolyzable in the cell.
- the present invention provides a method for facilitating the transfer of nucleic acids into cells.
- the method comprises preparing a mixed lipid dispersion of a cationic lipid with a co-lipid in a suitable carrier solvent, such as distilled water or normal saline solution.
- the cationic lipid has a structure which includes a lipophilic group derived from cholesterol, a linker bond, a spacer arm including from about 1 to about 20 carbon atoms in a branched or unbranched linear alkyl chain, and a cationic amino group.
- the amino group is selected from the group consisting of primary, secondary, tertiary and quaternary amino groups.
- the method further comprises adding the nucleic acids to the dispersion to form a complex.
- the cells are then treated with the complex.
- the dispersion has particles with an average diameter of about 150nm.
- the cationic lipid is preferentially selected from the group consisting of cholesteryl-3 ⁇ -carboxylamidoethylenetrimethylammonium iodide, 1-dimethylamino-3-trimethylammonio-DL-2-propyl-cholesteryl carboxylate iodide, cholesteryl-3 ⁇ -carboxyamidoethyleneamine, cholesteryl-3 ⁇ -oxysuccinamidoethylenetrimethylammonium iodide, 1-dimethylamino-3-trimethylammonio-DL-2-propylcholesteryl-3 ⁇ -oxysuccinate iodide, 2-[(2-trimethlyammonio)-ethylmethylamino] ethyl-cholesteryl-3 ⁇ -oxysuccinate iodide, 3 ⁇ [N-(N', N'-dimethylaminoethane)-carbamoyl]chol
- the present invention also provides a substantially non-toxic,substantially non-hydrolyzable cationic lipid for facilitating the transfer of nucleic acids into cells.
- the lipid comprises a lipophilic group derived from cholesterol, a linker bond, a spacer arm including from about 1 to about 20 carbon atoms in a branched or unbranched linear alkyl chain, and a cationic amino group.
- the amino group is selected from the group comprising primary, secondary, tertiary or quaternary amino groups.
- the cationic lipid is preferably selected from the group consisting of cholesteryl-3 ⁇ -carboxyamidoethylenetrimethylammonium iodide, 1-dimethylamino-3-trimethylammonio-DL-2-propyl-cholesteryl carboxylate iodide, cholesteryl-3 ⁇ -carboxyamidoethyleneamine, cholesteryl-3 ⁇ -oxysuccinamidoethylenetrimethylammonium iodide, 1-dimethylamino-3-trimethylammonio-DL-2-propyl-cholesteryl-3 ⁇ -oxysuccinate iodide, 2 - [ (2-trimethylammonio)ethylmethylamino]ethyl-cholesteryl-3 ⁇ -oxysuccinateiodide, 3 ⁇ [N-(N',N'dimethylaminoethane)-carbamoyl]-cholesterol, and 3 ⁇ [N- (polyethyleneimine)-carbamoyl]cholesterol
- FIGURE 1 A synthetic scheme for cholesteryl carboxylate analogues is shown in FIGURE 1.
- Cholesterol, (25 g, 64.6 mmol) was dissolved in 10 mL of dimethylaniline (78.9 mmol) and 5 mL of chloroform. While stirring on ice; small quantities of thionyl bromide (6 mL, 77.6 mmol) dissolved in 20 mL of cold chloroform was added slowly over a period of 15 minutes. After the addition of thionly bromide was complete, the mixture was stirred for an additional 2 hours at room temperature. The resulting solution was poured into 200 mL of ice cold 95% ethanol and left on ice for 2 hour until crystallization was complete. The product was filtered and washed with 25 mL of ice cold 95% ethanol.
- cholesteryl-3 ⁇ -carboxylate was performed using a Grignard reaction. All glassware was oven dried at 110°C overnight. In a 500 mL three-neck flask set up for reflux, a solution of methyl magnesium iodide was freshly prepared by treating 9 g of oven dried (110°C) magnesium powder in 100 mL anhydrous diethyl ether with 10 mL of methyl iodide. After the vigorous reaction subsided, cholesteryl bromide (25 g, 56 mmol) dissolved in 100 mL of anhydrous diethyl ether was slowly added to the methyl magnesium iodide solution over a three hour period.
- the solution was refluxed for 36 hours with enough heat required to bring the diethyl ether to a boil.
- the Grignard reagent was added to finely ground solid carbon dioxide, and after 1 hour, the complex was hydrolyzed by treatment with ice cold 1 M sulfuric acid.
- the steroid was extracted with diethyl ether (3 x 250 mL)
- the ethereal layer was washed with 10 mM sodium thiosulfate (3 x 50 mL) to remove a persistent orange color.
- the ether layer was washed with distilled water and filtered to remove an insoluble residue.
- acylation of cholesteryl carboxylate was carried out under a dry argon or nitrogen atmosphere in oven dried glassware.
- Cholesterol carboxylate (2 g, 4.8 mmol) was suspended in 5 mL of dichloromethane (HPLC grade under 4 ⁇ molecule sieves).
- a 1.5 molar excess of 1,1'-carbonyldiimidazole (CDI, 1.2 g) dissolved in 15 mL dichloromethane was added to the cholesteryl carboxylate suspension is small volumes with intermittent shaking. When the reaction subsided, the solution was stirred overnight. N,N-dimethylethylenediamine (5 mL, 43.2 mmol) was subsequently added and the resulting solution was stirred for 36 hours at room temperature.
- the quaternization of Compound III was performed using methyl iodide and potassium bicarbonate. Briefly, 1 g (2.1 mmol) of compound III was dissolved in 40 mL of methanol in the presence of 2 g (20 mmol) of potassium bicarbonate and 2 mL (32.1 mmol) of methyl iodide. The reaction was stirred for 24 hours at room temperature. The solvent was subsequently removed under vacuum and the remaining bicarbonate was neutralized with 1 M HCl until the solution gave a pH reading of 7. Water was removed by lyophilization and the product was extracted from inorganic salt impurities using a small volume of ice cold methanol.
- the product was recrystallized from absolute ethanol and was further purified on a reverse phase column using an acetonitrile/0.1% trifluoroacetic acid gradient (100% to 85% acetonitrile in 60 minutes).
- the product was characterized by proton NMR.
- the product had a molecular ion with an m/z of 557 (M +° ) with FAB+ mass spectroscopy, consistent with the alkylation of one of the possible two tertiary amine sites.
- the product was characterized by proton NMR.
- the chloroform phase was subsequently dried with anhydrous sodium sulfate, the solvent removed and the residue purified by preparative TLC using chloroform:methanol:water (65:25:4, v/v/v) as the developing solvent.
- Mass spectrometry (FAB+) gave an m/z of 457 for the protonated molecular ion (M+H)+.
- the product was characterized by proton NMR.
- FIGURE 2 A scheme describing the various steps for producing cholesterol hemisuccinate analogues is depicted in FIGURE 2.
- compound VIII first required the acyl imidazolide of cholesteryl hemisuccinate which was prepared by reacting cholesterol hemisuccinate with N,N-carbonyldiimidazole (CDI) as described for the synthesis of compound III. Briefly, to cholesterol hemisuccinate (2 g, 4.1 mmol) suspended in 5 mL of dichloromethane was added 1.5 equivalents of CDI (1 g) dissolved in 15 mL of dichloromethane. The solution was stirred overnight after which N,N-dimethylethylenediamine (5 mL, 43.2 mmol) was added.
- CDI N,N-dimethylethylenediamine
- Cationic cholesterol derivatives were mixed with a phospholipid in chloroform solution at different molar ratios.
- the solvent was removed by evaporation under a stream of N 2 gas and desiccated in vacuo for at least 30 minutes.
- the dry lipid film was hydrated in 20 mM Hopes buffer, Ph 7.8, overnight.
- Plasmid pUCSV2CAT (approximately 5kb in size) containing the structural gene of E. coli chloramphenicol acetyl transferase (CAT) driven by the SV40 virus early promoter was used as a model for the polyanions to be delivered by the cationic lipid dispersions.
- DNA was mixed with cationic lipid dispersions in 1 ml serum-free M199 medium or McCoy's medium to form DNA/lipid complex. Cultured mammalian cells of about 80-100% confluency in a 6-well plate were washed once with serum-free medium. The DNA/lipid complex was added to the washed cells which were incubated at 37°C for 5 hours.
- the cells were washed again and the serum-containing medium was added. Cells were harvested 30-72 hours later and extracted for cellular proteins.
- the CAT activity in the extracted protein was measured by using either [ 14 C] chloramphenicol or [ 3 H] acetyl CoA as a radiolabeled substrate.
- One activity unit of CAT is defined as nmole of radiolabeled substrate converted to the radiolabeled product in one minute. Protein content in the cell extracts was measured by the Bradford (BIORAD) assay.
- brains for 25 Sprague-Dawley rats were removed, washed with 100 mL of 20 mM TRIS, 1 mM EDTA, 1mM EGTA, Ph 7.5, and homogenized in 150 mL of ice cold 20 mM TRIS, 10 mM EGTA, 2 mM EDTA, 10 mM DTT, 0.25 M sucrose, 2 mM PMSF and 100 ⁇ g/mL leupeptin, pH 7.5.
- the homogenate was immediately centrifuged at 100,000 g for 40 minutes at 4° C in a Beckman Ti 50.2 rotor.
- the supernatant was applied to a 2.5 X 20 cm column of DEAE Sepharose (fast flow) containing 60 mL of resin equilibrated with 20 mM TRIS, 1 mM EDTA, 1 mM DTT, pH 7.5 (buffer A).
- the column was washed with 300 mL of buffer A and an additional 200 mL of buffer A containing 0.03 M KCl.
- Protein kinase C was eluted with a 500 mL continuous KCl gradient (0.03 - 0.3 M KCl). Fractions of 5 mL volumes were collected. Fractions showing calcium and phospholipid dependence were pooled; the salt concentration was adjusted to 1.5 KCl with the appropriate quantity of solid KCl.
- the crude sample containing 1.5 M KCl was stirred for 15 minutes and subsequently loaded onto a 1 X 10 cm column containing 9 mL Phenyl separose equilibrated with 1.5 M KCl in 20 mM TRIS, 0.5 mM EGTA, 1 mM DTT, pH 7.5 (buffer B).
- the column was washed with 90 mL of buffer B containing 1.5 M KCl.
- PKC was eluted with a 100 mL continuous KCl gradient (1.5 - 0 M KCl). Fractions of 3 mL volumes were collected.
- the column was washed with an additional 50 mL of buffer B. Most of the enzyme activity eluted during this stage.
- Fractions showing calcium and phospholipid dependence were pooled and concentrated to 4 mL using an Amicon ultrafiltration cell fitted with a YM-10 filter. The concentrated sample was loaded onto a 2.5 X 100 cm column containing 400 ml of Sephacryl S-200 HR beads equilibrated with buffer B containing 10% glycerod (buffer C). Fractions of 3 mL volumes were collected. About 150 mL of buffer was run through; PKC eluted very close to the column void volume. The fractions showing calcium and phospholipid dependence were pooled and loaded onto a 0.5 x 5 cm column containing 2.5 mL polylysine agarose equilibrated with buffer C.
- PKC was eluted with a 40 mL continuous KCl gradient (0-0.8 M KCl). Fractions of 1 mL volumes were collected. The first few active fractions were contaminated. The uncontaminated fractions were pooled, concentrated, and diluted with buffer C to remove the high salt content. After reconcentrating, the sample was divided into working portions, frozen in liquid nitrogen and stored at -80°C. Full activity was regained after rapid thawing. Trace impurities (116 k, 66 k, and 50 k Mr) could still be detected when the gel was silver stained heavily.
- the enzyme gave a specific activity of 200 nmoles phosphate incorporated per minute per milligram of protein when assayed for histone phosphorylation using the Triton mixed micelle assay with 6.5 mole % phosphatidylserine, 2.5 mole % DAG and 100 ⁇ M calcium present. Specific activities ranging from 30 nmoles/min/mg to 600 nmoles/min/mg have been observed for PKC using the Triton mixed micelle assay under the same conditions.
- Phosphatidylserine and 1,2-diolein with and without additive were dissolved in a solution of chloroform/methanol (2:1, v/v). Solvent was evaporated with a stream of nitrogen and last traces removed using a vacuum desiccator at 40°C. The lipid films were then solubilized by the addition of 3% Triton X-100, vortexed vigorously for 30 seconds and then incubated at 30°C for 10 minutes to allow for equilibration.
- Plasmid DNA pUCSV2CAT was used as a model compound for polyanions because it contains a structural gene for CAT.
- the efficiency of intracellular delivery can be readily assayed by the expression of CAT activity in the extracted proteins of the treated cells.
- Table 1 lists the CAT activity of mouse L929 cells which have been transfected with this plasmid DNA as mediated by various cationic lipid dispersions.
- Compound VII deserves some special attention. It contains only a single primary amino group with a short spacer arm, yet the transfection activity was relatively high.
- Example XXI The experiments described in Example XXI were done with a lipid dispersion containing a cationic cholesterol derivative and a co-lipid dioleoyl phosphatidylethanolamine (DOPE).
- DOPE co-lipid dioleoyl phosphatidylethanolamine
- FIGURE 4 shows the data of an experiment in which compound VIII was mixed with a variety of different co-lipid, neutral and acidic, at a molar ratio of 1:1. The DNA delivery activity of these mixed dispersions were then studied. As can be seen, only DOPE supported the delivery activity of compound VIII.
- Other neutral lipids such as dioleoyl phosphatidylcholine (DOPC), N-methyl-DOPE, N,N-dimethyl DOPE had little or no activity. None of the acidic lipids, such as PS and phosphatidylglycerol (PG) showed any activity.
- DOPC dioleoyl phosphatidylcholine
- PG phosphati
- FIGURE 5 shows that maximal DNA delivery activity of the dispersion occurred when the dispersion contained 20-50% compound VIII. Too much or too little of compound VIII in the mixed dispersion did not yield good delivery activity.
- FIGURE 6 shows the data of an experiment in which various amounts of dispersion were added to a fixed amount of DNA (5 ⁇ g) for transfection. Maximal activities occurred at 69-80 nmoles of dispersion. We then used 70 nmoles dispersion and varied the amount of DNA for transfection (Fig. 7). The bell-shaped curve in the figure indicates that a 5 ⁇ g DNA gave the maximal activity. Thus the optimal ratio of dispersion-to-DNA was 70 nmole lipid for 5 ⁇ g DNA.
- A431 human epidermoid carcinoma cells were used for the transfection experiments.
- a fixed amount of DNA (4 ⁇ g) was mixed with an increasing amount of cationic lipid dispersion or a commercially available transfection reagent, Lipofectin, and added to the A431 cells for transfection (FIGURE 9).
- the toxicity of the treatment to the cells was measured as the total amount of cellular protein extractable at the time of CAT activity assay.
- Lipofectin treated cells showed a greatly reduced protein content with 50% inhibition occurring at about 7 ⁇ g lipid/ml.
- Lipid dispersions were prepared with various cationic cholesterol derivatives and DOPE (about 1:1 molar ratio). The transfection activities of the dispersions were tested at different times after the dispersions were stored at 4°C in PBS, pH 7.5. Of the derivatives listed in Table I, only the dispersions containing compounds XIV and XV were stable after storage; their transfection activities did not change for at least 2 months. On the other hand, the dispersions composed of other derivatives lose activity after 2-3 days in storage. Compounds XIV and XV contain a carbamoyl linker bond whereas other compounds contain either an ester bond or an amide bond. It is known that ester and amide bonds are more sensitive than the carbamoyl bond to hydrolysis particularly in the presence of bases.
- the cationic derivatives may catalyze the hydrolysis of each other's ester bonds, leading to the inactivation of the delivery activity.
- Compounds containing carbamoyl linker bonds are less sensitive to the base-catalyzed hydrolysis, yet they can still be hydrolysed by cellular enzymes, i.e., they are biodegradable. This is in contrast to the non-degradable ether bond in DOTMA which is the active ingredient of Lipofectin.
- DOTMA non-degradable ether bond in DOTMA which is the active ingredient of Lipofectin.
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Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
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US751873 | 1991-08-28 | ||
US07/751,873 US5283185A (en) | 1991-08-28 | 1991-08-28 | Method for delivering nucleic acids into cells |
PCT/US1992/007290 WO1993005162A1 (en) | 1991-08-28 | 1992-08-28 | Method for delivering nucleic acids into cells |
Publications (3)
Publication Number | Publication Date |
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EP0663013A4 EP0663013A4 (en) | 1994-11-28 |
EP0663013A1 EP0663013A1 (en) | 1995-07-19 |
EP0663013B1 true EP0663013B1 (en) | 2005-04-06 |
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Application Number | Title | Priority Date | Filing Date |
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EP92920321A Expired - Lifetime EP0663013B1 (en) | 1991-08-28 | 1992-08-28 | Method for delivering nucleic acids into cells |
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US (1) | US5283185A (es) |
EP (1) | EP0663013B1 (es) |
JP (2) | JP3258661B2 (es) |
AT (1) | ATE292690T1 (es) |
AU (1) | AU665029B2 (es) |
CA (1) | CA2116676A1 (es) |
DE (1) | DE69233497T2 (es) |
DK (1) | DK0663013T3 (es) |
ES (1) | ES2240958T3 (es) |
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WO (1) | WO1993005162A1 (es) |
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JPH07500963A (ja) | 1995-02-02 |
WO1993005162A1 (en) | 1993-03-18 |
EP0663013A4 (en) | 1994-11-28 |
AU665029B2 (en) | 1995-12-14 |
SG50630A1 (en) | 1998-07-20 |
DE69233497D1 (de) | 2005-05-12 |
EP0663013A1 (en) | 1995-07-19 |
HK1014736A1 (en) | 1999-09-30 |
ATE292690T1 (de) | 2005-04-15 |
US5283185A (en) | 1994-02-01 |
JP3258661B2 (ja) | 2002-02-18 |
DE69233497T2 (de) | 2006-01-26 |
ES2240958T3 (es) | 2005-10-16 |
AU2656592A (en) | 1993-04-05 |
JP2002153270A (ja) | 2002-05-28 |
DK0663013T3 (da) | 2005-08-01 |
CA2116676A1 (en) | 1993-03-18 |
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